This invention relates to an improved in vitro process for assaying for the presence of endotoxin in a biological fluid including the blood of animals and parenteral fluids such as serum, plasma, whole blood, albumins, dextrose solutions, and the like.
Generally speaking, endotoxin is a complex lipopolysaccharide material derived from gram-negative bacilli that is known to produce a wide variety of striking pathophysiological reactions in animals. For many years it was believed that the material was contained within gram-negative bacilli cells and was released only upon disintegration of the cell walls. Hence, the material was termed endotoxin. Recent studies, however, have shown that endotoxin is localized at the cell surface of gram-negative bacilli and may be present with viable and killed cells as well as in a free from within a liquid medium.
Endotoxin has been identified as a direct and contributory cause of death of many hospitalized patients. More particularly, endotoxin is known to cause febrile reactions in animals with symptoms of extremely high fever, vasodilation, diarrhea, and the like and, in extreme cases, fatal shock. It is also known that endotoxin causes leucocytosis, deleterious changes in carbohydrate and protein metabolism and widespread intravascular clotting by fibrin formation.
Studies have shown that endotoxemia in animals may be caused by or is associated with gram-negative bacilli primary and secondary infections and/or the employment of intravenous apparatus or solutions contaminated with gram-negative bacilli or endotoxin. The occurrence of endotoxemia rom the use of endotoxin-contaminated intravenous or parenteral solutions has recently been recognized as a particular problem in modern hospitals. For these reasons, a considerable amount of research has been, and is presently being conducted, to develop a simple, rapid, positive process for detecting the presence of endotoxin in parenteral fluids and in the blood of animals.
There are several procedures known for detecting the presence of endotoxin in many types of biological fluids. More particularly, there are several known bioassay procedures for detecting endotoxin which take advantage of one or more of the biologic effects or the antigenic composition of endotoxin through the use of experimental animals. For example, one of the most widely accepted bioassays for endotoxin is the rabbit pyrogenicity test which is currently used to satisfy Federal Drug Administration requirements for parenteral solutions and biologic products designed for intravenous injection in man. The rabbit pyrogenicity test is carried out by injecting a biological fluid being tested into three or more preconditioned rabbits and continuously monitoring the rectal temperatures of the injected rabbits for at least 3 hours following the injection. Biological fluids contaminated with endotoxin cause febrile reactions in the test rabbits with increased rectal temperatures being observed. In addition, several bioassay procedures for detecting endotoxin have been described which utilize the lethal effects of endotoxin in test animals.
However, known bioassay procedures suffer from several disadvantages. Known bioassay procedures not only require extensive amounts of time but are also relatively difficult to perform. Moreover, such procedures have not been found to be particularly sensitive for detecting the presence of endotoxin and suffer in reliability in view of all the variabilities encountered through the required use of intact test animal systems.
Several in vitro assay procedures for detecting endotoxin in biological fluids are also known. For example, D. K. Heilman and R. C. Bast, Jr., in "J. Bacteriol." 93: 15-20 (1967), disclose an in vitro assay process for detecting endotoxin through the inhibition of macrophage migration by endotoxin. A process for determining the presence of endotoxin in parenteral fluids by the use of membrane filtration has been described by S. Marcus in "Bull. Parenteral Drug Ass." 18:18-24 (1964). However, studies have shown that such in vitro methods are no more sensitive in detecting the presence of endotoxin than many of the aforementioned bioassay procedures and exhibit no significant improvement in reliability.
Several in virto procedures have recently been described for the detection of endotoxin in certain biological fluids by the use of amebocyte lysate from the hemolymph of the horseshoe crab, Limulus polyphemus. Studies have shown that the amebocyte lysate of Limulus crabs is extremely sensitive to endotoxin and coagulates in its presence in a gelation reaction. It has been amply demonstrated that the degree of gelation or coagulation of the amebocyte lysate is quantitative to the amount of endotoxin present. Further, it has been found that the amebocyte lysate is so sensitive to endotoxin as to detect as little as 0.1 ng of endotoxin present in 1 ml of fluid.
Generally, in vitro procedures for detecting the presence of endotoxin in biological fluids employing the amebocyte lysate of Limulus crabs include admixing a sample of the biological fluid with the lysate, incubating the admixture for a predetermined period of time at a predetermined temperature, and observing and grading the degree of gelation of the lysate. Examples of particular procedures employing the Limulus lysate test for detecting endotoxin in a variety of parenteral fluids are described in the following publications: R. R. Rojas-Corona et al. "The Limulus Coagulation Test for Endotoxin: A Comparison with Other Assay Methods," Proc. Soc. Exp. Biol. Med. 132:599-601 (1969); J. F. Cooper et al. "Quantitative Comparison of In Vitro and In-Vivo Methods for the Detection of Endotoxin," J. Lab. Clin. Med. 78:138-147 (1971); and J. F. Cooper et al. "The Limulus Test for Endotoxin (Pyrogen) in Radiopharmaceutical and Biologicals," Bull. Parenteral Drug Ass. 26:153-162 (1972).
Processes have also been described employing the amebocyte lysate of Limulus crabs for detecting endotoxin in human blood. However, such known procedures require extensive treatment of the blood or blood components, such as plasma, prior to testing with the Limulus lysate to remove certain inhibitors that prevent the detection of endotoxin. For example, J. Levin et al. in "Detection of Endotoxin in Human Blood and Demonstration of an Inhibitor," J. Lab. Clin. Med. 75:903-911 (1970), describe a process for detecting endotoxin in human blood by the use of Limulus amebocyte lysate which includes initial treatment of blood plasma containing endotoxin with a 1:4 ratio of chloroform to plasma for a period of 1 hour to remove certain endotoxin inhibitors in the plasma prior to incubation with the Limulus lysate.
R. B. Reinhold et al, in "A Technique for Quantitative Measurement of Endotoxin in Human Plasma," Proc. Soc. Exp. Biol. Med. 137:334-340 (1970), describe the use of a pH shift method of plasma treatment to retard endotoxin binding by plasma proteins in an in vitro process for detecting endotoxin employing Limulus amebocyte lysate.
We have now discovered an improved in vitro process for detecting the presence of endotoxin in the blood of an animal by the use of amebocyte lysate of Limulus crabs which does not require extensive treatment of the blood or any of its components to remove certain plasma inhibitors that was heretofore required in known processes. The process of our invention can be carried out rapidly and simply with no adverse affect on the apparent sensitivity and specificity of Limulus amebocyte lysate to endotoxin. The process of the invention may also be employed for the detection of endotoxin in any type of biological fluid, including parenteral fluids.